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DNA encoding an arthropod chitin synthase
6465179 DNA encoding an arthropod chitin synthase
Patent Drawings:Drawing: 6465179-2    Drawing: 6465179-3    Drawing: 6465179-4    Drawing: 6465179-5    Drawing: 6465179-6    
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Inventor: Thireos, et al.
Date Issued: October 15, 2002
Application: 09/230,041
Filed: January 19, 1999
Inventors: Kafetzopoulos; Dimitris (Heraklion, GR)
Thireos; George (Heraklion, GR)
Assignee: Foundation for Research and Technology-Hellas (Heraklion/Crete, GR)
Primary Examiner: Prouty; Rebecca E.
Assistant Examiner: Hutson; Richard
Attorney Or Agent: McDermott, Will & Emery
U.S. Class: 435/193; 435/252.3; 435/320.1; 435/6; 435/69.1; 435/69.2; 536/23.2
Field Of Search: 435/193; 435/69.1; 435/69.2; 435/320.1; 435/6; 435/252.3; 536/23.2
International Class:
U.S Patent Documents: 5824545
Foreign Patent Documents:
Other References:









Abstract: The present invention relates to nucleic acids comprising a nucleotide sequence encoding at least a portion of an enzyme which catalyzes the synthesis of chitin in arthropods, inhibitors directed to said enzyme, and a method for developing said inhibitors.
Claim: What is claimed is:

1. An isolated nucleic acid which comprises a nucleotide sequence that hybridizes under 0.75 M Na.sup.+ and 55.degree. C. conditions to the DNA sequence shown in SEQ ID NO:1,wherein said nucleic acid encodes an enzyme which catalyzes the synthesis of chitin in arthropods.

2. The nucleic acid according to claim 1, wherein the nucleotide sequence contains the DNA sequence as shown in SEQ ID NO:1.

3. A recombinant vector comprising a nucleic acid according to claim 2.

4. The recombinant vector according to claim 3, wherein said nucleic acid sequence is functionally linked to an expression-control sequence.

5. A host containing the nucleic acid according to claim 2.

6. A method for developing chitin synthase inhibitors, comprising the steps of: (a) expressing the nucleic acid of claim 1 in a host cell, and (b) comparing chitinsynthase activity in the presence and absence of test compounds.

7. A host containing the recombinant vector according to claim 3.
Description: The present invention relates to nucleic acids comprising a nucleotide sequence encoding at least a portion of anenzyme which catalyzes the synthesis of chitin in arthropods, inhibitors directed to said enzyme, and a method for developing said inhibitors.

Chitin is a carbohydrate homopolymer of .beta.(1.fwdarw.4) linked N-acetylglucosamine. Chitin is a structural polysaccharide occurring mainly in the cell wall of some fungi and in exoskeleton of the arthropods.

Chitin is synthesized by the action of specialized enzymes, referred to as chitin synthases, that catalyze the polymerization of N-acetylglucosaminyl residues into chitin from uridine 5'-diphospho-N-acetylglucosamine.

Chitin synthases belong to a group of enzymes that catalyze the synthesis of linear structural polysaccharides and are collectively termed as processive glycosyltransferases. The primary structures of the known processive glycosyl transferases(i.e. the fungal chitin synthases, the prokaryotic and eukaryotic cellulose synthases, the prokaryotic and eukaryotic hyaluronan synthases, the prokaryotic alginate syntheses and the rhizobial chito-oligosaccharide syntheses) present some sequencesimilarities suggesting that these UDP-sugar utilizing enzymes show a similar mode of action. Despite the similarities, the various processive glucosyltransferases display substantial differences mainly in their substrate specificities, their activationand inhibition parameters, the size and conformation of their products.

Since several arthropod species are considered as plant and animal pests, the inhibition of the chitin synthesis machinery has been viewed in particular as an attractive target for the development of pest control agent.

Known insect chitin synthesis inhibitors, either natural or synthetic, fall within two main categories: (a) direct inhibitors of chitin synthesis such as the Streptomyces antibiotics Nikkomycins and Polyoxins and (b) insect growth regulators thatinterfere indirectly but drastically with the synthesis of chitin in insects, such us the benzoylphenyl-urea derivatives.

Although the studies for the identification of these compounds have a history of several decades, the action of chitin synthesis inhibitors is still inadequately understood. This is due to both the limited availability of suitable insect chitinsynthase enzyme preparations and to the lack of any molecular insight on the chitin synthesis and its regulation in insects.

Thus, the technical problem underlying the present invention is to provide new inhibitors for the chitin synthesis in order to prevent damages caused by arthropods.

The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.

In particular, there is provided a nucleic acid which comprises a nucleotide sequence encoding at least a portion of an enzyme which catalyzes the synthesis of chitin in arthropods.

The terms "nucleic acid" and "nucleotide sequence" refer to endogenously expressed, semisynthetic, synthetic or chemically modified acid molecules of deoxyribonucleotides and/or ribonucleotides. In a preferred embodiment of the present inventionthe nucleotide sequence include sequences as illustrated in FIGS. 13 (SEQ ID NO:1), allelic derivatives of said sequences and DNA-sequences degenerated as a result of the genetic code for said sequences. It also includes DNA sequences hybridizing understringent conditions with the nucleotide sequence defined above. Although said allelic, degenerate and hybridising sequences may have structural divergences due to (naturally occurring) mutations, such as small deletions or substitutions, they willusually still exhibit essentially the same useful properties, allowing their use in basically the same applications.

In a preferred embodiment the present invention relates to an isolated DNA sequence encoding a portion of an enzyme which catalyzes the synthesis of chitin in arthropods. Specific embodiments include DNA sequences which are characterized by theability to hybridize to the DNA sequence represented in FIG. 1 at e.g. 55.degree. C. or encode a polypeptide reactive with an antibody against the polypeptide containing the deduced amino acid sequence in FIGS. 1 to 3. The invention also relates to aDNA expression construct containing the isolated DNA sequences and encoding an enzyme having the specificity described above.

The present invention also relates to recombinant molecules comprising the nucleic acid as described above optionally linked to an expression-control sequence. Such vectors may be useful in the production of at least said portion of an enzyme instable or transiently transformed cells. Several animal, plant, fungal and bacterial systems may be used for the transformation and subsequent cultivation process. Preferably, expression vectors which can be used in the invention contain sequencesnecessary for the replication in the host cell and are autonomously replicable. It is also preferable to use vectors containing selectable marker genes which can be easily selected for transformed cells. The necessary preparation is well known to thoseskilled in the art.

It is another object of the invention to provide a host cell transformed by an expression plasmid of the invention and capable of producing at least said portion of an enzyme. Examples of suitable host cells include various eukaryotic andprokaryotic cells, such as E. coli, insect cells, plant cells, mammalian cells, and fungi such as yeast.

Another object of the present invention is to provide a polypeptide comprising at least a portion of an enzyme which catalyzes the synthesis of chitin in arthropods. The term "polypeptide" includes the complete enzyme or a biologically active orinactive fragment thereof encoded by the sequences described above and displaying preferably biologically features of said enzyme. The amino acid sequences of specially preferred polypeptides contain the sequences displayed in FIGS. 13 (SEQ ID NO:2).

It is a further aspect of the invention to provide a process for the production of the above mentioned polypeptide. Such a process comprises cultivating a host cell being transformed with a nucleic acid sequence of the present invention in asuitable culture medium and purifying the produced polypeptide. Thus, this process allows the production of the sufficient amount of the desired polypeptide for use in applications described herein below. The host cell is obtainable from bacteria suchas E. coli, from fungi, such as yeast, from plants such as tobacco, potato, or Arabidopsis, and from animals, in particular vertebrate cells such as the CHO-cells.

The invention also relates to the methods for assaying the effects of various compounds on the expression and the biological activity of arthropod chitin synthases. In these methods the polypeptide in enzymatically active form, preferably theenzyme chitin synthase of an arthropod is produced by recombinant DNA techniques in which an isolated DNA sequence encoding the enzyme is expressed from a DNA expression construct as outlined above.

The figures show:

FIGS. 1 to 3 show DNA sequence (SEQ ID NO:1) and the deduced amino acid sequence (SEQ ID NO:2) of a portion of chitin synthase of Drosophila melanogaster.

FIGS. 4 and 5 show multiple protein sequence alignments of the portion of chitin synthase of Drosophila melanogaster with the corresponding regions of a nematode, three yeast chitin syntheses and a rhizobial chito-oligosaccharide synthase. Inparticular, the multiple sequence alignments show the isolated portion of Drosophila melanogaster chitin synthase (Dmechs1 in FIG. 4, and Dmechs2 in FIG. 5) with the corresponding regions of the nematode chitin synthase (Celchs), the chitin synthase 1(Scechs1), the chitin synthase 2 (Scechs2) and chitin synthase 3 (Scechs3) of yeast and the rhizobial chito-oligosaccharide synthase (RlenodC). The dots above the alignment indicate similarity and the asterisks identity.

In the followingpreferred embodiments of the present invention are outlined in more detail.

With the isolation of the first insect chitin synthase disclosed here, that of Drosophila melanogaster (fruitfly), recombinant methodology for the expression, purification and identification of chitin synthases can be used. These methodsemployed in assays for the evaluation and characterization of insect chitin synthase inhibitors can lead to the development of new potent pest control agents.

The present invention was made possible with identification of the DNA sequence shown in FIGS. 1 to 3, encoding for a portion of chitin synthase of Drosophila melanogaster.

A search of the GENBANK databank using the deduced amino acid sequence of Drosophila melanogaster sequence revealed significant similarities with a nematode sequence presumably encoding for a chitin synthase, with several sequences encoding forfungal chitin syntheses, and with several rhizobial chito-oligosaccharide syntheses. The multiple sequence alignments of the Drosophila melanogaster chitin synthase with these sequences are presented in FIGS. 4 and 5.

Despite the sequence similarities of the Drosophila melanogaster chitin synthase with above described enzymes, hybridization of the Drosophila melanogaster DNA to neither the nematode nor the fungal genomic DNA could be observed even at lowstringency conditions (0.75M [Na.sup.+ ], 55.degree. C.).

With the isolation of the new insect chitin synthase gene, that of D. melanogaster, the use of recombinant methodology in the production, characterization and identification of arthropod chitin synthases can be employed.

By reference to the DNA sequence listed below, hybridization, immunochemical or polymerase chain reaction amplification methods can be used for the isolation of either cDNA or genomic DNA of chitin synthase from this species. The same approachescan be used for the identification and isolation of DNA encoding the chitin synthases of other arthropod species.

The isolated DNA sequences which fall within the scope of this invention can be used to express the encoded chitin synthases in large quantities in either prokaryotic or eukaryotic host cell.

In another aspect, the invention relates to methods for developing pest control agents. Chitin synthases of arthropods can be produced by recombinant DNA methodology as outlined above and used in enzyme assays for the evaluation andcharacterization of specific chitin synthase inhibitors. These preparations can also be used in the screening for the identification of new chitin synthesis inhibitors.

Further, DNA constructs of the arthropod chitin synthases fused with reporter genes (lacZ, GFP, luciferase) can be used to obtain trangenic insects or tranformed cell lines as outlined above. These animals and cell lines can be used in theevaluation of effect insect growth regulators have in the biosynthesis of chitin.

The present invention will be further illustrated by the following examples.

EXAMPLE 1

A DNA fragment containing the isolated DNA sequence and encoding for the insect chitin synthase is inserted in an expression vector containing a lac inducible promoter. The construct is used to transform E. coli cells. Cells are grown in LBmedium at half-log phase and the expression of chitin synthase is induced by the addition of 1 mM IPTG.

After 4 hours the cell are harvested by centrifugation and lysed by sonication. The homogenate is centrifuged at 2000 g for 30 minutes at 4.degree. C. and the supernatant of the centrifugation is ultracentrifuged at 100 000 g for one hour. Thepellet containing the membrane fraction were resuspended in a buffer containing 25 mM Tris HCl pH 7.0 and 10 mM Magnesium chloride. This membrane preparation is subsequently used in chitin synthase inhibition assays.

The assays are performed by incubating a sample of the membrane fraction with UDP-GlcNAc 5 mM and the compounds tested for chitin synthesis inhibition. The activity of chitin synthase is evaluated by either the measurement of incorporation ofradiolabelled GlcNAc from UDP-GlcNAc into chitin or by the spectrophotometric estimation of released UDP by the method of the coupled enzyme reactions assay. Comparison of the measured activity to that obtained under control conditions (no compoundadded) is used for the evaluation of compounds as a chitin synthase inhibitor.

EXAMPLE 2

A DNA fragment encoding the promoter and a portion of the coding regions of the insect chitin synthase is fused to lacZ reporter gene. The DNA construct containing the translational fusion of Chitin synthase-lacZ is used for the transformationof Kc insect cells. After the selection and isolation of stable tranformants the transformed cell line is used for the testing or screening for compound that affect the transcriptional activation of the chitin synthase gene. In these assays the cellline is incubated with the compounds for 24 hours and the effect of the compounds is evaluated by measuring and comparing the beta-galactosidase activity to that of the control.

EXAMPLE 3

100 ng of the DNA fragment, encoding the sequence presented in FIGS. 1-3 is used for the incorporation of radioactive nucleotides into the double-stranded DNA according the standard "nick translation" methodology. Radioactive DNA probesgenerated by this method are used for the identification and isolation of clones encoding for chitin syntheses from a genomic DNA cosmid library of D. melanogaster. The DNA of the recombinant cosmids is placed (blotted) on nitrocellulose filters,denatured and thin hybridized with the radioactive DNA probe. Hybridisation is performed at low stringency conditions (0.75M [Na.sup.+ ], 55.degree. C.). Filters are then washed and exposed for autoradiography.

Cosmids which hybridized with the probe (positive clones), are isolated and further characterized with restriction enzyme analysis and DNA sequence analysis.

From the procedure described above a second gene (Dmechs2) encoding for chitin synthase in this species is identified and partially sequences. The DNA sequence and the deduced amino acid sequence are presented in FIGS. 1-3.

FIG. 5 shows multiple protein sequence alignment of the portion of Dmechs2 with the corresponding regions of a nematode, three yeast chitin syntheses and a rhizobial chito-oligosaccharide synthase.

SEQUENCE LISTING <100> GENERAL INFORMATION: <160> NUMBER OF SEQ ID NOS: 42 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 1 <211> LENGTH: 810 <212> TYPE: DNA <213> ORGANISM: Drosophilamelanogaster <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(810) <400> SEQUENCE: 1 gcc att gga cac tgg ctg caa aag gcg acg gag cat gtc att ggt tgc 48 Ala Ile Gly His Trp Leu Gln Lys Ala Thr Glu His Val Ile Gly Cys 1 5 10 15 gtc ctc tgc agt ccg gga tgc ttc agt ctg ttc cgg gcc acc gcc ctg 96 Val Leu Cys Ser Pro Gly Cys Phe Ser Leu Phe Arg Ala Thr Ala Leu 20 25 30 atg gag aac agc gtg atg aag cgg tac acg atg atc agt tcg gag gcg 144 Met Glu Asn Ser Val Met Lys ArgTyr Thr Met Ile Ser Ser Glu Ala 35 40 45 atg aag atg gtg cag tac gac cag ggc gag gac cgc tgg ctg tgt acg 192 Met Lys Met Val Gln Tyr Asp Gln Gly Glu Asp Arg Trp Leu Cys Thr 50 55 60 ctg ctc ttg aag gcc ggc ttc cgg gtg gag tac tgg ggc gcc ttg gat 240 Leu Leu Leu Lys Ala Gly Phe Arg Val Glu Tyr Trp Gly Ala Leu Asp 65 70 75 80 ggg tat acc caa gcg cca gag agc ttt aac gag ttc tac aac caa cga 288 Gly Tyr Thr Gln Ala Pro Glu Ser Phe Asn Glu Phe Tyr Asn Gln Arg 85 90 95 agt cgt tgg atc gcc att gga cactgg ctg caa aag gcg acg gag cat 336 Ser Arg Trp Ile Ala Ile Gly His Trp Leu Gln Lys Ala Thr Glu His 100 105 110 gtc att ggt tgc gtc ctc tgc agt ccg gga tgc ttc agt ctg ttc cgg 384 Val Ile Gly Cys Val Leu Cys Ser Pro Gly Cys Phe Ser Leu Phe Arg 115120 125 gcc acc gcc ctg atg gag aac agc gtg atg aag cgg tac acg atg atc 432 Ala Thr Ala Leu Met Glu Asn Ser Val Met Lys Arg Tyr Thr Met Ile 130 135 140 agt tcg gag gcg atg aag atg gtg cag tac gac cag ggc gag gac cgc 480 Ser Ser Glu Ala Met Lys MetVal Gln Tyr Asp Gln Gly Glu Asp Arg 145 150 155 160 tgg ctg tgt acg ctg ctc ttg aag gcc ggc ttc cgg gtg gag tac tgc 528 Trp Leu Cys Thr Leu Leu Leu Lys Ala Gly Phe Arg Val Glu Tyr Cys 165 170 175 ggc gcc tcg gat ggg tat acc cac gcg cca gag agc tttaac gag ttc 576 Gly Ala Ser Asp Gly Tyr Thr His Ala Pro Glu Ser Phe Asn Glu Phe 180 185 190 tac aac caa cga cgc cgt tgg atc gcc ctc atg gat gac aat gtg atg 624 Tyr Asn Gln Arg Arg Arg Trp Ile Ala Leu Met Asp Asp Asn Val Met 195 200 205 aag aaa tacacg aca cgg tcg gat gag gct cgt cac tac gtg cag tac 672 Lys Lys Tyr Thr Thr Arg Ser Asp Glu Ala Arg His Tyr Val Gln Tyr 210 215 220 gat cag ggc gag gat cgt tgg ctg tgc aca ttg ctc ctc cag agg gga 720 Asp Gln Gly Glu Asp Arg Trp Leu Cys Thr Leu LeuLeu Gln Arg Gly 225 230 235 240 tac cgc gtg gag tac tcg gct gcc agt gat gcg tac acc cac gcc ccc 768 Tyr Arg Val Glu Tyr Ser Ala Ala Ser Asp Ala Tyr Thr His Ala Pro 245 250 255 gag gga ttc aat gag ttc tac aac cag cgg cgg aga tgg gtg 810 Glu Gly PheAsn Glu Phe Tyr Asn Gln Arg Arg Arg Trp Val 260 265 270 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211> LENGTH: 270 <212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 2 Ala Ile GlyHis Trp Leu Gln Lys Ala Thr Glu His Val Ile Gly Cys 1 5 10 15 Val Leu Cys Ser Pro Gly Cys Phe Ser Leu Phe Arg Ala Thr Ala Leu 20 25 30 Met Glu Asn Ser Val Met Lys Arg Tyr Thr Met Ile Ser Ser Glu Ala 35 40 45 Met Lys Met Val Gln Tyr Asp Gln Gly GluAsp Arg Trp Leu Cys Thr 50 55 60 Leu Leu Leu Lys Ala Gly Phe Arg Val Glu Tyr Trp Gly Ala Leu Asp 65 70 75 80 Gly Tyr Thr Gln Ala Pro Glu Ser Phe Asn Glu Phe Tyr Asn Gln Arg 85 90 95 Ser Arg Trp Ile Ala Ile Gly His Trp Leu Gln Lys Ala Thr Glu His 100 105 110 Val Ile Gly Cys Val Leu Cys Ser Pro Gly Cys Phe Ser Leu Phe Arg 115 120 125 Ala Thr Ala Leu Met Glu Asn Ser Val Met Lys Arg Tyr Thr Met Ile 130 135 140 Ser Ser Glu Ala Met Lys Met Val Gln Tyr Asp Gln Gly Glu Asp Arg 145 150 155 160 TrpLeu Cys Thr Leu Leu Leu Lys Ala Gly Phe Arg Val Glu Tyr Cys 165 170 175 Gly Ala Ser Asp Gly Tyr Thr His Ala Pro Glu Ser Phe Asn Glu Phe 180 185 190 Tyr Asn Gln Arg Arg Arg Trp Ile Ala Leu Met Asp Asp Asn Val Met 195 200 205 Lys Lys Tyr Thr Thr ArgSer Asp Glu Ala Arg His Tyr Val Gln Tyr 210 215 220 Asp Gln Gly Glu Asp Arg Trp Leu Cys Thr Leu Leu Leu Gln Arg Gly 225 230 235 240 Tyr Arg Val Glu Tyr Ser Ala Ala Ser Asp Ala Tyr Thr His Ala Pro 245 250 255 Glu Gly Phe Asn Glu Phe Tyr Asn Gln ArgArg Arg Trp Val 260 265 270 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 3 Ala Ile Gly His Trp Leu Gln Lys Ala ThrGlu His Val Ile Gly Cys 1 5 10 15 Val Leu Cys Ser Pro Gly Cys Phe Ser Leu Phe Arg 20 25 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 4 Ala Thr Ala Leu Met Glu Asn Ser Val Met Lys Arg Tyr Thr Met Ile 1 5 10 15 Ser Ser Glu Ala Met Lys Met Val Gln 20 25 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 5 Gln Gly Glu Asp Arg Trp Leu Cys 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Drosophilamelanogaster <400> SEQUENCE: 6 Thr Leu Leu Leu Lys 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 7 AlaGly Phe Arg Val Glu Tyr Trp Gly Ala Leu Asp Gly Tyr Thr Gln 1 5 10 15 Ala Pro Glu Ser Phe Asn Glu Phe Tyr Asn Gln Arg Ser Arg Trp Ile 20 25 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 23 <212> TYPE:PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 8 Ala Leu Met Asp Asp Asn Val Met Lys Lys Tyr Thr Thr Arg Ser Asp 1 5 10 15 Glu Ala Arg His Tyr Val Gln 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 9 Gln Gly Glu Asp Arg Trp Leu Cys 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 10 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 10 Thr Leu Leu Leu Gln 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 11 <211> LENGTH: 32 <212> TYPE: PRT <213>ORGANISM: Drosophila melanogaster <400> SEQUENCE: 11 Arg Gly Tyr Arg Val Glu Tyr Ser Ala Ala Ser Asp Ala Tyr Thr His 1 5 10 15 Ala Pro Glu Gly Phe Asn Glu Phe Tyr Asn Gln Arg Arg Arg Trp Val 20 25 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 12 <211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 12 Ala Ile Ala His Trp Phe Gln Lys Ala Ala Glu His Val Phe Gly Cys 1 5 10 15 Val Leu Cys Ala Pro Gly Cys PheSer Leu Phe Arg 20 25 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 13 <211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 13 Ala Ser Ala Leu Met Asp Asp Asn Ile Met HisLys Tyr Thr Lys Thr 1 5 10 15 Ala Ser Glu Pro Arg His Tyr Val Gln 20 25 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 14 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE:14 Gln Gly Glu Asp Arg Trp Leu Ser 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 15 Thr Leu Leu Leu Lys 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 16 <211> LENGTH: 63 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 16 Gln Gly Tyr Arg Ile Glu Tyr Ala Ala Ala Ser Asp Ala Glu Thr Tyr 1 510 15 Ala Pro Glu Gly Phe Glu Glu Phe Phe Asn Gln Arg Arg Arg Trp Thr 20 25 30 Ala Leu Met Asp Asp Asn Ile Met His Lys Tyr Thr Lys Thr Ala Ser 35 40 45 Glu Pro Arg His Tyr Val Gln Tyr Asp Gln Gly Glu Tyr Val Gln 50 55 60 <200> SEQUENCECHARACTERISTICS: <210> SEQ ID NO 17 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 17 Gln Gly Glu Asp Arg Trp Leu Ser 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQID NO 18 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 18 Thr Leu Leu Leu Lys 1 5

<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 19 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 19 Gln Gly Tyr Arg Ile Glu Tyr Ala Ala Ala Ser Asp Ala Glu ThrTyr 1 5 10 15 Ala Pro Glu Gly Phe Glu Glu Phe Phe Asn Gln Arg Arg Arg Trp Thr 20 25 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 20 <211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 20 Lys Met Ser Asn Ile Leu Asp Lys Thr Thr Glu Ser Asn Phe Gly Phe 1 5 10 15 Ile Thr Val Leu Pro Gly Ala Phe Ser Ala Tyr Arg Phe Glu Ala Val 20 25 30 Arg Gly Gln 35 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO21 <211> LENGTH: 71 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 21 Leu Gln Lys Tyr Phe Tyr Gly Glu Ile Met Glu Asn Glu Gly Phe His 1 5 10 15 Phe Phe Ser Ser Asn Met Tyr Leu Ala Glu Asp Arg IleLeu Cys Phe 20 25 30 Glu Val Val Thr Lys Lys Asn Cys Asn Trp Ile Leu Lys Tyr Cys Arg 35 40 45 Ser Ser Tyr Ala Ser Thr Asp Val Pro Glu Arg Val Pro Glu Phe Ile 50 55 60 Leu Gln Arg Arg Arg Trp Leu 65 70 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 22 <211> LENGTH: 71 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 22 Leu Gln Lys Tyr Phe Tyr Gly Glu Ile Met Glu Asn Glu Gly Phe His 1 5 10 15 Phe Phe Ser Ser Asn Met Tyr LeuAla Glu Asp Arg Ile Leu Cys Phe 20 25 30 Glu Val Val Thr Lys Lys Asn Cys Asn Trp Ile Leu Lys Tyr Cys Arg 35 40 45 Ser Ser Tyr Ala Ser Thr Asp Val Pro Glu Arg Val Pro Glu Phe Ile 50 55 60 Leu Gln Arg Arg Arg Trp Leu 65 70 <200> SEQUENCECHARACTERISTICS: <210> SEQ ID NO 23 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 23 Lys Ile Ser Asn Ile Leu Asp Lys Pro Leu Glu Ser Val Phe Gly Tyr 1 5 10 15 Ile Ser ValLeu Pro Gly Ala Leu Ser Ala Tyr Arg Tyr Arg Ala Leu 20 25 30 Lys Asn His Glu Asp Gly Thr Gly Pro 35 40 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 24 <211> LENGTH: 71 <212> TYPE: PRT <213> ORGANISM: Saccharomycescerevisiae <400> SEQUENCE: 24 Leu Arg Ser Tyr Phe Leu Gly Glu Thr Gln Glu Gly Arg Asp His Asp 1 5 10 15 Val Phe Thr Ala Asn Met Tyr Leu Ala Glu Asp Arg Ile Leu Cys Trp 20 25 30 Glu Leu Val Ala Lys Arg Asp Ala Lys Trp Val Leu Lys Tyr Val Lys 35 40 45 Glu Ala Thr Gly Glu Thr Asp Val Pro Glu Asp Val Ser Glu Phe Ile 50 55 60 Ser Gln Arg Arg Arg Trp Leu 65 70 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 25 <211> LENGTH: 71 <212> TYPE: PRT <213> ORGANISM:Saccharomyces cerevisiae <400> SEQUENCE: 25 Leu Arg Ser Tyr Phe Leu Gly Glu Thr Gln Glu Gly Arg Asp His Asp 1 5 10 15 Val Phe Thr Ala Asn Met Tyr Leu Ala Glu Asp Arg Ile Leu Cys Trp 20 25 30 Glu Leu Val Ala Lys Arg Asp Ala Lys Trp Val Leu LysTyr Val Lys 35 40 45 Glu Ala Thr Gly Glu Thr Asp Val Pro Glu Asp Val Ser Glu Phe Ile 50 55 60 Ser Gln Arg Arg Arg Trp Leu 65 70 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 26 <211> LENGTH: 67 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 26 Tyr Ile Ser His His Gln Ala Lys Ala Phe Glu Ser Val Phe Gly Ser 1 5 10 15 Val Thr Cys Leu Pro Gly Cys Phe Ser Met Tyr Arg Ile Lys Ser Pro 20 25 30 Lys Gly Ser Asp Gly Tyr TrpVal Pro Val Leu Ala Asn Pro Asp Ile 35 40 45 Val Glu Arg Tyr Ser Asp Asn Val Thr Asn Thr Leu His Lys Lys Asn 50 55 60 Leu Leu Leu 65 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 27 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 27 Leu Gly Glu Asp Arg Phe Leu Ser 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 28 <211> LENGTH: 65 <212> TYPE: PRT <213> ORGANISM:Saccharomyces cerevisiae <400> SEQUENCE: 28 Ser Leu Met Leu Lys Thr Phe Pro Lys Arg Lys Gln Val Phe Val Pro 1 5 10 15 Lys Ala Ala Cys Lys Thr Ile Ala Pro Asp Lys Phe Lys Val Leu Leu 20 25 30 Ser Gln Arg Arg Arg Trp Ile Val Leu Ala Asn Pro AspIle Val Glu 35 40 45 Arg Tyr Ser Asp Asn Val Thr Asn Thr Leu His Lys Lys Asn Leu Leu 50 55 60 Leu 65 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 29 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Saccharomycescerevisiae <400> SEQUENCE: 29 Leu Gly Glu Asp Arg Phe Leu Ser 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 30 <211> LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400>SEQUENCE: 30 Ser Leu Met Leu Lys Thr Phe Pro Lys Arg Lys Gln Val Phe Val Pro 1 5 10 15 Lys Ala Ala Cys Lys Thr Ile Ala Pro Asp Lys Phe Lys Val Leu Leu 20 25 30 Ser Gln Arg Arg Arg Trp Ile 35 <200> SEQUENCE CHARACTERISTICS: <210> SEQID NO 31 <211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Rhizobium leguminosarum <400> SEQUENCE: 31 Trp Leu Ala Cys Asn Glu Glu Arg Ala Ala Gln Ala Arg Phe Gly Ala 1 5 10 15 Val Met Cys Cys Cys Gly Pro Cys Ala Met Tyr Arg 20 25 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 32 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Rhizobium leguminosarum <400> SEQUENCE: 32 Arg Ser Ala Met Leu 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 33 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Rhizobium leguminosarum <400> SEQUENCE: 33 Ser Leu Leu Asp Gln Tyr 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 34 <211>LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Rhizobium leguminosarum <400> SEQUENCE: 34 Glu Thr Gln Leu Tyr Arg Gly Lys Pro 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 35 <211> LENGTH: 8 <212> TYPE:PRT <213> ORGANISM: Rhizobium leguminosarum <400> SEQUENCE: 35 Phe Gly Glu Asp Arg His Leu Thr 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 36 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:Rhizobium leguminosarum <400> SEQUENCE: 36 Ile Leu Met Leu Ser 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 37 <211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Rhizobium leguminosarum <400>SEQUENCE: 37 Ala Gly Phe Arg Thr Glu Tyr Val Pro Ser Ala Ile Ala Ala Thr Val 1 5 10 15 Val Pro Asp Thr Met Gly Val Tyr Leu Arg Gln Gln Leu Arg Trp Ala 20 25 30 Ala Met Leu 35 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 38 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Rhizobium leguminosarum <400> SEQUENCE: 38 Ser Leu Leu Asp Gln Tyr 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 39 <211> LENGTH: 9 <212> TYPE:PRT <213> ORGANISM: Rhizobium leguminosarum <400> SEQUENCE: 39 Glu Thr Gln Leu Tyr Arg Gly Lys Pro 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 40 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:Rhizobium leguminosarum <400> SEQUENCE: 40 Phe Gly Glu Asp Arg His Leu Thr 1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 41 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Rhizobium leguminosarum <400> SEQUENCE: 41 Ile Leu Met Leu Ser

1 5 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 42 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Rhizobium leguminosarum <400> SEQUENCE: 42 Ala Gly Phe Arg Thr Glu Tyr Val Pro Ser Ala Ile Ala AlaThr Val 1 5 10 15 Val Pro Asp Thr Met Gly Val Tyr Leu Arg Gln Gln Leu Arg Trp Ala 20 25 30

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